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Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies.
J Virol. 2014 Dec; 88(24):14002-16.JV

Abstract

The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4(+) CCR5(+) host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies. Here we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7, and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G, and 10G cores. Following purification, most glycoproteins, except for 10G, were recognized by broadly neutralizing CD4bs-directed antibodies. Gel and glycan mass spectrometry confirmed that additional N-glycans were posttranslationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low to no binding by non-broadly neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar in their neutralization characteristics to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera, and the antisera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared to be trimer dependent. Fine mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface of Env sites of vulnerability to neutralizing antibody, suggesting an explanation for why the elicited neutralization was not improved by this rational design strategy.

IMPORTANCE

Major obstacles to developing an effective HIV-1 vaccine include the variability of the envelope surface glycoproteins and its high-density glycan shield, generated by incorporation of host (human) glycosylation. HIV-1 does harbor highly conserved sites on the exposed envelope protein surface of gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan masking. We used the atomic-level structure to reengineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to mask (by glycan) nonneutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of the modified gp120 core.

Authors+Show Affiliations

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.IAVI Neutralizing Antibody Center at TSRI, La Jolla, California, USA.Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.Laboratory of Viral Diseases, NIAID, NIH, Bethesda, Maryland, USA.Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA.Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA IAVI Neutralizing Antibody Center at TSRI, La Jolla, California, USA.Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA.Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA.Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA IAVI Neutralizing Antibody Center at TSRI, La Jolla, California, USA wyatt@scripps.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25253346

Citation

Ingale, Jidnyasa, et al. "Hyperglycosylated Stable Core Immunogens Designed to Present the CD4 Binding Site Are Preferentially Recognized By Broadly Neutralizing Antibodies." Journal of Virology, vol. 88, no. 24, 2014, pp. 14002-16.
Ingale J, Tran K, Kong L, et al. Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies. J Virol. 2014;88(24):14002-16.
Ingale, J., Tran, K., Kong, L., Dey, B., McKee, K., Schief, W., Kwong, P. D., Mascola, J. R., & Wyatt, R. T. (2014). Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies. Journal of Virology, 88(24), 14002-16. https://doi.org/10.1128/JVI.02614-14
Ingale J, et al. Hyperglycosylated Stable Core Immunogens Designed to Present the CD4 Binding Site Are Preferentially Recognized By Broadly Neutralizing Antibodies. J Virol. 2014;88(24):14002-16. PubMed PMID: 25253346.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies. AU - Ingale,Jidnyasa, AU - Tran,Karen, AU - Kong,Leopold, AU - Dey,Barna, AU - McKee,Krisha, AU - Schief,William, AU - Kwong,Peter D, AU - Mascola,John R, AU - Wyatt,Richard T, Y1 - 2014/09/24/ PY - 2014/9/26/entrez PY - 2014/9/26/pubmed PY - 2015/1/22/medline SP - 14002 EP - 16 JF - Journal of virology JO - J Virol VL - 88 IS - 24 N2 - UNLABELLED: The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4(+) CCR5(+) host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies. Here we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7, and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G, and 10G cores. Following purification, most glycoproteins, except for 10G, were recognized by broadly neutralizing CD4bs-directed antibodies. Gel and glycan mass spectrometry confirmed that additional N-glycans were posttranslationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low to no binding by non-broadly neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar in their neutralization characteristics to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera, and the antisera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared to be trimer dependent. Fine mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface of Env sites of vulnerability to neutralizing antibody, suggesting an explanation for why the elicited neutralization was not improved by this rational design strategy. IMPORTANCE: Major obstacles to developing an effective HIV-1 vaccine include the variability of the envelope surface glycoproteins and its high-density glycan shield, generated by incorporation of host (human) glycosylation. HIV-1 does harbor highly conserved sites on the exposed envelope protein surface of gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan masking. We used the atomic-level structure to reengineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to mask (by glycan) nonneutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of the modified gp120 core. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/25253346/Hyperglycosylated_stable_core_immunogens_designed_to_present_the_CD4_binding_site_are_preferentially_recognized_by_broadly_neutralizing_antibodies_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=25253346 DB - PRIME DP - Unbound Medicine ER -